# Hydrogen sulfide promotes proliferation and regeneration of human cerebral microvascular endothelial cells via the sonic hedgehog signaling pathway

## TL;DR

Hydrogen sulfide helps brain blood vessel cells grow and recover under low oxygen by activating the SHH signaling pathway.

## Contribution

This study reveals a novel regulatory mechanism where H2S promotes endothelial cell regeneration via the SHH pathway under hypoxia.

## Key findings

- H2S and SHH increase VEGF and P-ERK1/2 while reducing apoptosis in HCMEC/D3 cells.
- H2S production by HMC3 cells is regulated by CBS and SHH proteins during hypoxia.
- Inhibiting SHH does not affect H2S levels, but CBS inhibition reduces both H2S and SHH.

## Abstract

We investigated the effects of hydrogen sulfide (H2S) and sonic hedgehog (SHH) on the proliferation, autophagy, and apoptosis of human microvascular endothelial cells (HCMEC/D3). We also explored the regulatory relationship between cystathionine-β-synthase (CBS) and the SHH pathway. Human microglia cells (HMC3) were stimulated under hypoxia to secrete H2S and SHH proteins, which were then co-cultured with HCMEC/D3 cells. The relationship between H2S and SHH was investigated by inhibiting the CBS or SHH pathways. Vascular endothelial growth factor (VEGF) and H2S levels were detected using ELISA. The mRNA and Protein levels of VEGF, Beclin-1, light chain 3 (LC3), Cysteine aspartic acid protease-3(caspase-3), CBS, SHH, extracellular regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 (P-ERK1/2) were determined by RT-PCR and western blot. The results indicated that H2S secretion by HMC3 increased during hypoxia, with both CBS and SHH proteins being up-regulated. The inhibition of CBS resulted in decreased levels of H2S and SHH in HMC3. When the SHH pathway is inhibited, H2S secretion levels remain unaffected. H2S and SHH proteins increased VEGF, P-ERK1/2, Beclin-1, and LC3 expression while reducing caspase-3 expression in HCMEC/D3 cells. H2S secretion by HMC3 promotes the proliferation and regeneration of HCMEC/D3 by regulating SHH protein and alleviating hypoxic injury.